Dietary· supplements of antioxidants reduce hprt mutant frequency in splenocytes of aging mice

Genetic

ELSEVIER

DNAging Instability

and Agmg

Mutation Research 33X (19%) 77-86

Dietaryssupplements of antioxidants reduce hprt mutant frequency in splenocytes of aging mice A.I. Gaziev ii. *, A.Ja. Podlutsky “, B.M. Panfilov

a, R. Bradbury

b

a Institute of Theorcticul and Experimental Biophysics. 142292 Pushchino, Moscow Region, Russia h Aeifvm Scientific Corporufion, Scuttle, WA 31083, USA Accepted 15 May 1995

Abstract The level of spontaneous and gamma-radiation-induced mutations in the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus as well as the decrease in frequency of these mutations in mice of various age pretreated with dietary supplements of an antioxidant mixture (vitamins C, E, beta-carotene, rutin, selenium, zinc) were studied in splenocytes of young (8-14-week-old) and aged (102-llO-week-old) male C57BL/6 mice. The frequency of spontaneous mutations in splenocytes of 102-llO-week-oid mice was higher by 68-W% than that in mice aged 8-14 weeks. On gamma-irradiation (0.5-5.0 Gy) of mice, the frequency of radiation-induced mutations (Vf assay) in aged mice was 2.3 to 3.6 times (depending on dose) higher than in young ones. Daily supplements of an antioxidant mixture to the diet of mice prior to irradiation showed an antimutagenic effect. The values of mutant frequency reduction factor (MFRF) for 14-llO-week-old mice fed with dietary antioxidants during 6 weeks prior to gamma-irradiation with doses of 2.0 and 5.0 Gy were 5.4 and 3.7, respectively. The frequency of radiation-induced mutations prevented or not prevented by antioxidants was much higher in aged mice than in young ones. Keywords: hprt mutation:

Ageing; Dietary antioxidant: Mouse splenocyte:Gamma-irradiation

1. Introduction

The DNA lesions occur despite the fact that the cells possess molecular defense systems. Among them are the ‘antioxidant’ enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase, which neutralize highly reactive oxygen radicals and hydroperoxides (Halliwel and Gutteridge, 1989; Pigeolet et al., 1990). The protection of DNA and other cellular macromoIecules against the action of free radicals is also provided by the antioxidants most of which enter the organism with the food (Catler, 1985; Borec, 1986; Hayatsu et al., 1988; Poot, 1991;

Factors of endogeneous and exogeneous nature constantly induce lesions in DNA and other macromolecules of an organism (Ames, 1984, 1989; Hanawalt, 1987; Mullaart et al., 1990). These lesions contribute to disturbances in the functioning of macromolecules and are assumed to play an important role in senescenceprocesses and various pathological states (Ames et al., 1993). * Corresponding

author. Fax: (709.5) 924 WY3

0921-8734/95/$09.50 CC‘ 1995 SSDI OY21-X733(9S~OOOl~-5

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Rousseu et al., 1992; Chart. 1993; Bettger, 1993). Another molecular system of protection is DNA repair which interferes with the accumulation of DNA lesions in the cell (Hanawalt. 1987; Borh et al., 1987; LindahI? 1990). Nevertheless, the experimental evidence and clinical observations show that various types of damage are accumulated in the cellular DNA of aged organisms (Ames, 1989; Fraga et al., 1990; Mullaart et al.. 1990; Ames et al.> 1993) and the frequency of spontaneous chromosomal and genetic somatic mutations grows with age (Crowley and Curis. 1963: Morley et al., 1982; Trainor et al., 1984: Martin et al., 1985; Fenech and Morley, 1986; Albertini et al., 1990; Nisitani et al.. 1990: Slagboom et al., 1991). Comparative analysis of the structural DNA lesions arising spontaneously as the organism grows older and the DNA lesions found in organisms exposed to ionizing radiation shows a great similarity between the spectra of these alterations (Mullaart et al., 1YYtl; Teoule. 1987). Moreover, the radiobiological studies indicate that increasing the level of protectors-antioxidants in an organism under irradiation results in a reduction of DNA damage and mutation frequency and increases the radioresistance of the organism (Gebhart. 1992; Maisin, 1902). These data make us believe that the accumulation of DNA lesions and the increased somatic mutations in aging organisms may be due to a decrease in activity of the molecular defense systems. However, the studies concerned with the activity of the systems for neutralization of free radicals in aging organisms show ambiguous results (Cutler, 1985; Poot, 1991). As for the age-related changes in activity of the DNA repair enzymes, their intrinsic activity levels seem to he sufficient for the spontaneous DNA lesions to be repaired. but the access of these enzymes to DNA lesions within chromatin may be decreased by aging (Hanawalt, 1987; Lindahl, 1990; Mullart et al., 1990). On the other hand, many of the numerous antioxidants, vitamins and micronutrients applied at present as a means for chemoprevention of tumors or for suppression of development of some chronic diseases (Meyskens and Prasad, 1986; De Flora. and Romel, 1988; Diplock, 1991; Bronzetti et al., 1992) can increase the activity of defense systems in the

organism. When scavenging free radicals, they, probably, reduce the number of DNA lesions and modification of proteins in the cell (Kuroda, 1990). The present study is an attempt to determine the effect of dietary antioxidant supplements: beta-carotene. alpha-tocopherol, ascorbic acid, rutin as well as microelements selenium and zinc on the frequency of spontaneous and y-radiation-induced mutations at the hprt locus in mice of various age. The choice of this combined prescription was governed by the fact that the components of this formula taken together or in various combinations had been tested as dietary microsupplements capable of reducing the consequences of genotoxic and radiational stresses (Hayatsy et al., 1988; Bronzetti et al., 1992; Byers and Perry, 1992; Kelloff et al., 1992; Bettger, 1993; Davison et al., 1993; Sammon, 1993). The components of this prescription have a capacity to increase the activity of the organism’s defense systems and to inhibit mutagenesis in vitro and to decrease the risk of carcinogenesis and the development of some other pathologies (Meyskens and Prazad. 1986; Simic and Bergtold, 1991; Kelloff et al.. 1992: Byers and Perry, 1992). Also, both the experimental and clinico-epidemiological observations show that beta-carotene, vitamins, and other compounds used in chemoprevention of tumors exhibit a particular organic and tissue specificity (Meyskens and Prazad, 1986; Bronzetti et al.. 1992; Kelloff et al., 1992). Moreover, some antioxidants and vitamins must be introduced in a combination to prevent their disbalance in the organism (Xu et al., 1992; Chan, 1993). Rutin, selenium, and zinc increase the general antioxidant reserves in the cells by different mechanisms. Besides, zinc may substitute copper and iron ions as well as participate in the activation of numerous Zn-dependent enzymes and in the induction of metallothioneins (Bray and Bettger, 1990; Bremner and Beattie, 1990; Coleman, 1992; Bettger, 1993; Sammon, 1993). Our experimental results showed that daily supplements of a mixture of vitamins C, E, rutin, and beta-carotene as well as selenium and zinc to the diet of g-110- week-old mice for 6 weeks decreased the frequency of mutant splenocytes at

the hprt locus after gamma-irradiation of these mice. In all cases the frequency of hprt mutations in splenocytes was much higher in aged mice than in young ones.

2. Materials

and methods

2.1. Animals and dietary supplements antioxidant mixture

of an

Male C57BL/6 mice purchased from the nursery of the Laboratory of Experimental Models. Russia Academy of Medical Sciences, were used. The mice were kept under standard conditions with a daily light/darkness cycle 12 h: 12 h. They were given a standard commercial diet, and water ad libitum. Experimental groups of animals were additionally given an antioxidant mixture containing beta-carotene, vitamins C, E, rutin, as well as selenium and zinc within granules of fat-free curd. The control groups of mice received placebogranules of curd without the antioxidant mixture. Each mouse ate daily from 9-11 a.m.: 0.2 g of curd granules without or with the antioxidant microsupplements at doses calculated per kg body weight: beta-carotene, 7.5 mg; alpha-tocopherolacetate, 15 mg; ascorbic acid, 50 mg; sodium selenite, 25 pg; zinc gluconate, 38.4 mg (5 mg of elementary zinc) tall reagents were from Twin Lab., Ronkonkoma, New York); rutin (quercetin3P-rutinoside). 25 mg (Sigma, St. Louis). 2.2. Irradiation Mice and splenocyte suspensions were irradiated with y-rays on a “‘Cs installation at a fixed dose rate of 80 cGy/min. Dosimetry was provided by the ferrous sulfate method according to ICRU (1984). 2.3. Preparation

of splenocytes

Mice 8 weeks after irradiation and nonirradiated ones were killed by cervical dislocation. Isolation of splenocytes was performed according to procedures described by Gocke et al. (1983) and Dempsey and Morley (1986). The concentration

of viable cells was determined in aliquots by staining with tripan blue in a hemocytometer. The cells were then resuspended in the RPM1 1640 medium supplemented with 20% of FBS and 10% of DMSO (Sigma) at a concentration of 5 . 10h cells/ml and freezed in liquid nitrogen at a rate of l”C/min. After 1 to 5 days, the cells were thawed out and washed in the RPM1 1640 culture medium by centrifugation, resuspended again, and the cell density was adjusted to be 1 . 10” cells/ml. 2.4. Autoradiographic and clonal assays for quantibing hprt-mutant cells Basic assays for determining 6-thioguanine-resistant (TG’) cells were carried out autoradiographically (variant frequency (Vf)-assay) (Albertini et al., 1982, 1988; Gocke et al., 1983) but for comparison a clonal assay (mutant frequency (Mf)-assay) was also used (Dempsey and Morley, 1986; Albertini et al., 1988; Grdina et al., 1992). In autoradiographic assays, after thawing out 1 . 10” cells were incubated in 1 ml of the RPM1 1640 incubation medium supplemented with: 12% FBS 25 mM Hepes, 2 mM L-glutamine, 100 units/ml penicillin, 100 pg/ml streptomycin, 5. lops M 2-mercaptoethanol, 0.075% sodium bicarbonate, 5 pg/ml PHA-p and 30 units/ml human recombinant interleukin-2 thrIL-2). Cell suspension was introduced to 24-microwell plates (1 ml to each well). Additionally, 0.1 ml of RPM1 1640 medium with 2.5 pi/ml h-thioguanine (6TG) (selective medium) or without 6-TG were added to each well and the plates were incubated in a 5% CO,/95% air atmosphere at 37°C for 30h. Then, 0.1 ml of the RPM1 1640 medium containing 5 PCi [“HI thymidine (specific activity 40 Ci/mM) was added to each well, and the incubation was continued further for 16 h. The cell suspension was quantitatively transferred to 10 ml cone-shaped glass tubes. diluted with 0.14 M NaCl to a final volume of 7 ml, centrifuged and resuspended in 3 ml of fixative (ethanol glacial acetic acid, 3 : 1) and centrifuged once more. The pellets of the cells incubated in the selective medium were resuspended in 250 ~1 of fixative whereas the pellets from the 6-TG-free

culture were resuspended in 10 ml of fixative. The whole volume (25 ~1) of the first cell suspension and 25 ~1 portions of the second cell suspension were transferred onto microscope slides (18 x 18 mm). The slides were air dried, stained with Giemsa and dipped in Kodak NTP-2 photoemulsion at 45°C. The exposure was carried out for 4 days at 2-3°C. The number of cells with labeled nuclei per 1 . 106 cells was counted with due regard for dilutions and the frequency of TG’ variant (Vf) was estimated as described by Gocke et al (1983). In order to conduct the clonal assay, 1 . 10” cells were incubated in 1 ml of a culture medium containing all the supplements indicated above without or with 6-TG (2.5 pg/ml) for 40 h. The cells were then resuspended in a fresh culture medium but without PHA. After being diluted, the cell suspensionswere transferred to roundbottomed wells of 96well microtiter plates. In this case, one 0.1 ml portion of suspension of cells incubated without 6-TG contained 5 cells. whereas the same portion of cells incubated in the selective medium with 6-TG contained 5 . 10J cells. Also, 0.1 ml suspension of feeder cells resuspended in the RPM1 1640 culture medium (5 . 10’ mice splenocytes gamma-irradiated at a dose of 30 Gy) was introduced to each well. The plates were incubated for 14 days at 37°C in a humid atmosphere (5% CO2/95% air). The cloning efficiencies were calculated from the proportion of negative wells by using Poisson statistics, PO= e-“, as indicated by Albertini et al. (1988). The mutation frequency, was quantified as a ratio of cloning efficiency in the presence of 6-TG to cloning efficiency in the absenceof 6-TG. The experimental data were treated statistically (Beyer, 1966).

3. Results 3.1. Estimatiorz of background hprt mutations hi two different assays The results of comparative analysis of background hprt mutations in splenocytes of mice of various age are presented in Table 1. The data

Table I Frequency of hprt-mutations in splenocytes of mice of various age as determined by the cloning technique (Mf-assay) and autoradiography (Vf-assay) in h-TG-radioresistant cells Method

Mf-assay Vf-assay

14.week-old

mice

102-week-old

mice

No. of mice

Frequency (x10-6)

No. of mice

Frequency (x 10-h)

8 10

1.9 * 0.4 4.3 k 0.6

6 8

3.2 f 0.6 7.85 1.1

obtained by the autoradiographic assay(Vf assay) show increased values compared to those of the splenocyte cloning technique (Mf assay). This difference took place in assaying the hprt mutation frequency in splenocytes of both young and aged (102-week-old) mice. The results obtained by both assays show the background hprt mutation frequency in splenocytes of 102-week-old mice to be substantially higher compared to that for 14week-old mice. For instance, the mutation frequency quantified by lymphocyte cloning assay in aged mice was by 68% higher than that in young mice. The autoradiographic assays showed the frequency of spontaneous mutations in lymphocytes of aged mice to be by 81% higher than in young mice. 3.2. Radiation-induced hprt mutations in mice of r’arious age Table 2 presents the results of assaying Vf in h-TG-resistant splenocytes of &week- and 104week-old mice exposed to gamma-rays. In this

Table 2 Mutation week-old

frequency mice after

(Vf-assay) y-irradiation

in splenocytes

mice

of 8- and 104.

Irradiation dose (gy)

ii-week-old

104.week-old

mice

Frequency (x lo-“)

Degree of increase

Frequency (x lo-“)

Degree of increase

0 0.5 1.o 7.0 5.0

4.2*0X 8.2 + 0.7 14.8k 1.3 24.3 + 1.8 45.2 k 2.4

1.9 3.5 5.7 10.7

7.9* 1.4 2X3-1 4.1 44.5* 9.9 69.4+ 13.2 102.8k 12.8

2.8 5.6 8.7 13.0

Assays were carried out 56 days after each experiment S-6 mice were used.

irradiation

of mice.

In

Table 3 Mutation frequency Wf) in splenocytes of y-irradiated after receiving an antioxidant mixture with the diet Time of giving antioxidants (days) 0 3

Mutation frequency x lKh Wf) Irradiated mice

4.4io.7

x.3* 20.2 + 15.4-t 0.2 i s.0 i 4.x i

14 28 42

4.0*

7

MFRF

Nonirradiated mice 0.6

mice

1.x 2.1 1.7

2.0 0.4 0.7

L/

1.2 1.6 2.6 4.8

5.0

R-week-old mice received daily antioxidants. then they were y-irradiated (2 Gy), and 56 days after irradiation the mutation frequency was assayed; 6 mice were used for each experiment. “ MFRF: mutation frequency reduction factor. ratio between Vf of mice not given antioxtdants and that of mice received antioxidants

table, as well as in other tables and figures, the age of mice is indicated for the moment of irradiation. In order to obtain splenocytes. the animals were killed at day 56 after gamma-irradiation. The data presented in Table 2 and other tables were obtained by assaying 6-TG-resistant cells by autoradiography (Vf assay). The presented evidence indicates (Table 2) that the mutation frequency in splenocytes of irradiated mice increases as a function of irradiation dose. Within a dose range from 0.5 to 5.0 Gy. the production of hprt mutations in splenocytes shows an apparent linear dose dependence. This Table 4 Effect of dietary supplements of an antioxidant splenocvtes of various age mice . Mouse weeks

age.

Nonirradiated

mice

Frequency x 10 h

14 42 78

110

Placebo b

4.0 i 0.5 5.4 * 1.0 6.4 + 1.3

4.2 * 0.6

Animals were given for each experiment.

6.7 t 0.x 7.9 * 1.4

3.3. The antimutagenic effect of an antioxidant mtiture added to the diet of mice The increased level of background and radiation-induced mutations in splenocytes of 104week-old mice compared to that in young mice is probably the result of weakening of the molecular defense systems in the cells of aged animals. In order to assess the antimutagenic effect of the dietary supplements of antioxidant mixture, the mice were fed with curd granules containing antioxidants (the above-mentioned vitamins, betacarotene, rutin, selenium, zinc) for various time periods. The experimental results are presented in Table 3. It is seen that feeding mice with the antioxidant mixture did not readily result in any

on the frequency

of spontaneous

Mice irradiated with 2.0 Gy MFRF b/a

Antioxidant a

mixture

dose dependence is clearly seen for both 8-week and 104-week-old animals. At the same time, the mutation frequency in splenocytes of 104-weekold mice was much higher than that in 8-week-old mice for all irradiation doses. The data (Table 2) show that this increase is by no means the result of the contribution of the difference in background mutation level between splenocytes of young and aged mice only. Thus, the background mutation level in splenocytes of aged mice (Table 2) exceeds that in young mice only by 880/o, whereas the mutation frequency in cells of gamma-irradiated lObweek-old mice exceeds that in gamma-irradiated 8-week-old mice by 127 to 200% (depending on irradiation dose).

1.1 1.2 1.2

antioxidants during 6 weeks. MFRF. mutation frequency

MFRF h/a

Antioxidant a

Placebo b

4.6 s.x 11.4 IS.1

24.x

0,s I.0 2.4 4.2

The age of mice reduction factor.

induced

mutations

Mice irradiated with 5.0 Gy

Frequency x10 h

t + i t

and radiation

+ 39.Y j49.4 * 67.6 * is given h/a.

Antioxidant a 1.2 1.4 8.1 5.7

5.4

5.1 4.3 4.4

for the moment

MFRF

Frequency x lomh

8.7 + 0.7 9.9 * 3.8

18.8 * 7.1 i 7.2

28.2

of irradiation.

b/a Placebo b 46.8 * 2.1 49.7 + 5.1 75.2 f 11.4 104.9 f 15.3 6-8

mice were

5.3 5.0 4.0 3.7

analyzed

in

x2

Al.

Gazirr

et al. /Mtrtution

marked antimutagenic effect. A significant decrease in the frequency of radiation-induced mutations was observed beginning with 7 days of giving the antioxidant mixture. The maximum antimutagenic effect was reached for 4-week- and 6-week-periods of giving mice the antioxidant mixture, the mutation frequency reduction factors (MFRF) being 5.0 and 4.8, respectively. 3.4. Antimutagenic effect of the antioxidant mixture on mice of rsarious ages Mice of various age (14, 42. 78, and 110 weeks) received daily dietary supplements of an antioxidant mixture for 6 weeks, then the mice were gamma-irradiated, and 8 weeks later the Vf was measured in splenocytes. The experimental and control values are given in Table 4. It is seen that in nonirradiated 78- and IlO-week-old mice fed with antioxidants, the frequency of spontaneous mutations in splenocytes is only slightly decreased, the MFRFs being 1.24 and 1.23, respectively. The antimutagenic effect of the antioxidant mixture is much more pronounced in gamma-irradiated mice. This effect is seen in all age groups after irradiation with doses of 2.0 and 5.0 Gy. Thus, MFRFs in mice of 14 to IlO-weeks age are between 5.4 and 4.4 for the dose 2.0 Gy and between 5.3 and 3.7 for 5.0 Gy. The data presented in Table 4 point to some lowering of the antimutagenic effect of the antioxidant mixture with animal age. For instance, the MFRF in 14-week-old mice are 5.4 and 5.3 for irradiation doses of 2.0 and 5.0 Gy, whereas MFRF values obtained on 1 lo-week-old mice are 4.4 and 3.7 for the same doses. The aged mice exhibited a higher level of mutations not prevented by antioxidants than young mice do. In general, the frequency of radiation-induced mutations, both prevened and not prevented by antioxidants. was much higher in aged mice than in young ones. 4. Discussion As known, there are two basic methods (not counting their modifications) to determine the hprt mutation frequency in human and animal

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3.M 11995) 77-86

cells by counting 6-TG-resistant cells: (1) the clonogenic assay (Mf assay) and (2) the autoradiography of 6-TG-resistant cells (Vf assay) (Albertini et al., 1982, 1988, 1990). At the beginning of our study we used both assays to quantify spontaneous hprt mutations in splenocytes of 14-week- and 102-week-old mice. The frequency of spontaneous mutations quantified by the Mf assay in splenocytes of 14-week-old mice was (1.9 + 0.8) X 10eh. which is in agreement with literature data (Jones et al., 1985; Dempsey and Morley, 1986; Grdina et al., 1992). The Vf value of spontaneous mutations for the same cells determined by autoradiography was two times as high as the Mf value but much lower than the Vf value obtained with the same assay by Gocke et al., (1983). In any case, the frequency of spontaneous mutations, as determined by both Mf and Vf assays, increased with animal’s age. Thus, in 102 to IlO-week old mice, the spontaneous mutation frequency was by 68-88% higher than in young (8- to l4-week-old) mice. In subsequent experiments we used only the autoradiographic assay in quantifying hprt mutation frequency since it is more convenient for monitoring analyses (Albertini et al., 1988; 1990). Earlier it has been shown that the background hprt mutation frequency in human lymphocytes increases with age (Morley et al., 1982; Trainor et al., 1984; Dempsey et al., 1993). On the average, this increase for human peripheral blood lymphocytes is about 2% per year (Albertini, 1990; Mendelsohn, 1992). As for the mice, our experimental evidence indicates that the hprt mutation frequency in mouse splenocytes increases by 6888% within two years. According to our observations and the literature data (Zapadnuk et al., 19831, the lifetime of male C57BL/6 mice is 800 to 830 days. Probably, the significant increase in background mutation frequency in 102-l loweek-old (714-770 days) mice is due to the high specific rate of metabolic processes in mice, unlike those in man, and, respectively, to an increased rate of development of ‘metabolic’ promutagenic lesions in mouse cell DNA (Simic, 1992). However, the high background frequency of somatic mutations in aged cells may be the result of decreased activity of the molecular de-

fense systems in these cells: enzymes of radical and peroxide detoxication, cellular antioxidants, the enzymatic systems of DNA repair etc. The changes in integral activity of the molecular defense systems of cells in aged organisms can be judged from the increased frequency of mutations or DNA lesions induced by ionizing radiation or oxidative agents (Ames, 1984: Ames et al.. 1993: Cutler, 1985; Bohrn et al., 1987; Simic, 1988: Bronzetti et al., 1992; Hanawalt et al., 1992; De Flora and Ramel, 1988). Analysis of the literature data shows that the spectrum of DNA lesions induced by ionizing radiation and oxidative agents and the spectrum of ‘spontaneous’ DNA lesions occurring due to aging are qualitatively very similar (Teoule, 1987; Mullaart et al., 1990). We detected a higher yield of hprt mutations in splenocytes of gamma-irradiated aged mice as compared to those in irradiated young mice. We analyzed the mutation frequency levels in mice 56 days after irradiation, since considerable time is required for the maximum expression of hprt-mutations in splenocytes of irradiated mice (Jones, 1989; Grdina et al., 1992). The high level of hprt mutation frequency in the splenocytes of irradiated aged mice may really indicate a decrease in activity of the systems for protection of the genome against the radicals generated by gamma-radiation. Our experimental data on the antimutagenic effect of the antioxidant mixture given to mice as a dietary supplement also point to that the systems of genome protection decrease their activity upon animal’s aging. They are also in agreement with the suggestion that the decrease in activity of the cellular systems for detoxication of radicals is connected with organism’s aging (Culter, 1985; Pigeolet et al., 1990; Poot, 1991). If we really believe that the high frequency of spontaneous and radiation-induced mutations in the cells of aged mice is due to the decreased activity of the complex of molecular defense systems, then the frequency of such mutations may be expected to be reduced in animals receiving particular substances for increasing or supplementing the activity of defence systems in the organism. To test this suggestion, we used a complex antioxidant mixture as a supplement to a

standard mouse diet. All components of this mixture: beta-carotene, vitamins C, E, rutin, selenium, zinc are natural regulators of the cellular defense systems (Meyskens and Prasad, 1986; Hayatsu et al., 1988; Bronzetti et al., 1992). All the components of the antioxidant mixture were used in moderate doses, as distinct from the high doses of vitamins C and E reported by El-Nahas et al. (1993). On daily single feeding of mice with the antioxidant mixture, the antimutagenic effect of the latter was manifested beginning with the 7th day of feeding. This seems to be associated with accumulation in the splenocytes of these mice of antioxidants in ammounts sufficient for the defense system to be activated. Though this complex antioxidant preparation produced only a minor effect on the spontaneous mutation frequency in splenocytes of aged mice. The low decrease in spontaneous mutation frequency in aged mice produced by antioxidants may be due to that the spontaneous mutagenic DNA lesions are accumulated during a much greater time interval compared to that of giving dietary supplements, and the introduced antioxidants probably could prevent only those of mutations accumulated in the latter months before killing of mice. The assay of mutations affecting the hprt locus has been used as a genetic marker in studying the antimutagenic activity of various substances in animal and human cell cultures. In particular, vitamins A, C, E have been shown to decrease the frequency of 6-TG-resistant mutations induced by various mutagens (review, Kuroda et al., 1992). Aminothyol radioprotectors have capability of decreasing the frequency of hprt mutations induced by ionizing radiation in mammalian cells. For instance, aminothyol WR-2721 exerted an antimutagenic effect, as shown by hprt mutation assay, in splenocytes of mice exposed in vivo to ionizing radiation (Grdina et al., 1992; Kataoka et al., 1992). The antimutagenic effect of this aminothyol seemsto be connected with its ability to capture free radiacals, to decrease the intracellular oxygen and to promote DNA repair (Smoluk et al., 1988; Maisin, 1992). Probably, the mechanisms of the antimutagenic effect of the antioxidant mixture are partially the same as that of

x4

il. I. Gazkr

rt al. /Mutation

aminothyols, though the components of this mixture may have specific effects on the cellular metabolism. Our experimental evidence indicates that in splenocytes of aged mice that received the antioxidant mixture the radiation-induced mutations were prevented much stronger (in absolute values) than those in young mice (Table 4). At the same time, the residual level of radiation-induced mutations not prevented by antioxidants was also higher in splenocytes of 1lo-week-old mice in comparison to that in 14-week-old mice. It is likely that the increased frequency of hprt mutations not prevented by antioxidants in splenocytes of gamma-irradiated aged mice is due to the lesions occurring in the DNA sites within chromatin inaccessible for the antioxidant protection or for the repair enzymes. As known, the supramolecular organization of DNA within chromatin and its folding play an essential role in providing the access of various agents to DNA sites and in the efficiency of DNA repair (Bohr et al., 1987; Hanawalt et al., 1992). Disturbances in the structural organization of chromatin and in DNA folding may occur in the process of senescence due to formation of cross-linkages between protein molecules or between protein and DNA (Tas and Walford, 1982; Almager and Cole, 1989: Maciera-Coelho, 19911, and these disturbances within the chromatin structure may affect the regulation of enzymatic repair of DNA damage. Thus, our experimental results make us believe that in the cellular DNA of aged mice compared to that of young ones a greater number of promutagenic lesions are produced because of the decrease in activity of the molecular defense systems, and the mutation frequency in splenocytes of mice seems to be conditioned by the level of unrepaired DNA lesions. We succeeded to increase substantially the activity of these defense systems by introduction of an antioxidant mixture to the diet of mice, which is evidenced by the decrease in frequency of radiation-induced mutations in splenocytes of these mice. Acknowledgements

The authors thank 1. Smetanich and Juliya Nikolaeva for their valuable technical assistance.

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We are grateful to Margaret Il’ina for her excellent help in the preparation of the manuscript. This study was supported by the Aeiveos Corporation, Seattle, WA, 98103-50 20, USA.

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